Metastatic disease is the primary cause of cancer mortality, but effective treatments remain elusive. Therefore, our LONG-TERM GOAL is to address the CRITICAL NEED for more targeted strategies to inhibit metastatic cancer. Metastatic cancer cells migrate away from the primary tumor and invade the extracellular matrix to enter the circulatory system and establish secondary tumors at distant sites. Accordingly, this proposal, which is an extension of an ARRA supplement to SC3 GM084824 to study the role of Rho GTPases in breast cancer progression via regulation of cancer cell migration, will test novel anti metastatic cancer therapeutics. The Rho GTPases, Rac and Cdc42, are key molecular switches activated by a myriad of cell surface receptors to promote cancer cell migration/invasion, proliferation, and survival. However, there is a GAP IN KNOWLEDGE on the efficacy of Rac and Cdc42 inhibitors as anti metastatic cancer therapeutics. The OBJECTIVE in this proposal is to characterize small molecule compounds that block the interaction of Rac and/or Cdc42 with their upstream effectors, guanine nucleotide exchange factors (GEFs) in metastatic cancer. The RATIONALE for this research comes from our characterization of the Rac inhibitor EHop-016 and the Rac/Cdc42 inhibitor MBQ-167 in cancer cell and mouse models. EHop-016 and the improved compound MBQ-167 inhibit cancer cell migration/invasion, viability, and tumor growth, metastasis, and angiogenesis in mouse models with no apparent toxicity. Using rational drug design principles garnered from our studies with these Rac/Cdc42 inhibitors, the PI's collaborators synthesized a panel of MBQ-167 derivatives (50 compounds). The present proposal will use 6 of these compounds to test the HYPOTHESIS that MBQ-167 and analogs have potential as anti metastatic breast cancer therapeutics. SPECIFIC AIM 1 will elucidate the mechanism of action of MBQ-167 and analogs in non-metastatic and metastatic cancer cell lines, and a normal mammary epithelial cell line. EHop-016 inhibits the activation of Rac by the oncogenic GEF Vav, but the GEFs that interact with MBQ-167 remain to be identified. Therefore, this Aim will use novel targeted mass spectrometric and protein array approaches to identify the GEFs that interact with MBQ-167 and analogs to inhibit Rac and Cdc42 activities. SPECIFIC AIM 2 will determine the efficacy and toxicity of MBQ-167 in immunocompromised and immunocompetent mouse models. Successful completion of this study will uniquely advance our understanding of metastatic cancer by establishing Rac and Cdc42 proteins as viable targets to impede metastatic breast cancer progression. This study will also result in identification of the variation in GEF expression and activity in non-metastatic and metastatic cancers and elucidate targeted therapeutic strategies for our panel of inhibitors. Moreover, these novel chemical probes will be available as tools to validate Rac/Cdc42 signaling as well as the function of specific GEFs in disease models.! Ultimately, this study will IMPACT the development of metastasis-targeted therapies for breast cancer. !